PURIFICATION AND PROPERTIES OF CYTOCHROMES C OF AZOTOBACTER VINELANDII

1. 1. An improved method for extraction and purification of Azotobacter vinelandii cytochromes c by chromatography with DEAE-cellulose and Sephadex and by isoelectric focusing is described. Yields of purified cytochromes c4 and c5 were approximately double those obtained with previous methods. Cytochromes c4 and c5 were homogeneous as evidenced by their purity index values and electrophoretic patterns on acrylamide gel. Both were slowly autoxidizable; cytochrome c4 did not combine with CO, whereas cytochrome c5 showed 10% combination. 2. 2. A third cytochrome c from A. vinelandii, designated as minor cytochrome c4, was separated on DEAE-cellulose columns. Though spectrally identical, its molecular weight is half that of cytochrome c4. 3. 3. Cytochrome c4 is a monomer with 2 hemes per molecule and a molecular weight of 24000 ± 2000. Cytochrome c5 was isolated as a dimer of 24400 ± 1000 molecular weight, but under denaturing conditions it is split into monomers half this size, each containing 1 heme group. 4. 4. Cytochrome c4 has 4 histidines per molecule; cytochrome c5 has 1 histidine per monomer. Tryptophan is absent from cytochrome c4. 5. 5. Neither the relative cytochrome c content of cells grown on ammonia nor functional tests supported a direct role for A. vinelandii cytochromes c in nitrogen fixation. Large differences in rates of oxidation among the bacterial cytochromes c were noted with oxidase from A. vinelandii and from beef heart. Cytochrome c oxidation by A. vinelandii was always highly sensitive to KCN; this supports the concept that all bacterial cytochromes c are on one branch of a split terminal oxidase chain. © 1969.